CD141+ dendritic cells produce prominent amounts of IFN-α after dsRNA recognition and can be targeted via DEC-205 in humanized mice

Abstract

Functional differences between human dendritic cell (DC) subsets and the potential benefits of targeting them with vaccines remain poorly defined. Here we describe that mice with reconstituted human immune system components (huNSG mice) develop all human conventional and plasmacytoid DC compartments in lymphoid organs. Testing different Toll-like receptor agonists for DC maturation in vivo, we found that IL-12p70 and interferon (IFN)-α production correlated with the maturation of CD141+ (BDCA3+) conventional DCs in huNSG mice. Furthermore, depletion of CD141+ DCs before stimulation significantly reduced IFN-α levels in vivo. This DC subset produced similar total amounts but different subtypes of IFN-α in response to synthetic double-stranded RNA compared with plasmacytoid DCs in response to a single-stranded RNA equivalent. Moreover, synthetic double-stranded RNA as adjuvant and antigen targeting to the endocytic receptor DEC-205, a combination that focuses antigen presentation for T-cell priming on CD141+ DCs, stimulated antigen-specific human CD4+ T-cell responses. Thus, the human CD141+ DC subset is a prominent source of IFN-α and interleukin-12 production and should be further evaluated for vaccine development.

Figures

Figure 1

Human DC subsets in huNSG mice. (A) Flow cytometric staining of CD11c−, CD303+ pDCs, or CD1c+ and CD141+ on CD11c+ cDCs of the spleen (left). Samples were pregated as singlet, live cells positive for hCD45 and HLA-DR and negative for lineage, CD14, and CD16. Percentages of those subsets in relation to live, singlet, human CD45+ cells are shown in the middle. The numbers on top of the data points indicate the average percentage of human CD45-positive cells for each analyzed DC subset. Absolute numbers of DC populations are shown on the right. Graphs represent data from mice from 4 independent reconstitutions. Each data point represents one individually analyzed mouse. (B) Same as in panel A for DC subsets of the BM. (C) Same as in panel A for DC subsets of the blood. (D) Immunofluoresence microscopy for DC markers on spleen sections. DEC-205 in red as DC-marker, CD20 in green as B-cell marker, and CD3 in blue as T cell marker.

Figure 2

Maturation of human DC subsets upon TLR ligand injection in vivo. (A) Fold up-regulation of CD86, CD83, CD274, CD40, and HLA-DR on CD1c+ cDCs (top), CD303+ pDCs (middle), and CD141+ cDCs (bottom). HuNSG mice were injected intraperitoneally with 50 μg/mouse polyICLC, 20 μg/mouse GLA IDC 1001, 25 μg/mouse protamine/RNA, 20 μg/mouse R848, and 50 μg/mouse CpG ODN 2216 or PBS. At 14 hours after injection, splenocytes were isolated and stained for flow cytometry. Fold up-regulation was calculated from the mean fluorescence intensity (MFI) in relation to the mean of the corresponding PBS samples. The graph represents composite data from 5 independent experiments. Each data point represents one individually analyzed mouse. Statistical analysis was performed with the Mann-Whitney U test. (B) Immunohistochemistry for DC markers on spleen sections. HuNSG mice were injected with PBS (top) or with polyICLC (bottom) and euthanized after 14 hours. DEC-205 serves as a DC marker and DC-LAMP and HLA-DR as DC maturation markers.

Figure 3

Kinetics of human DC maturation and cytokine production in vivo. (A) Time-course for the up-regulation of maturation markers CD86 and CD83 on splenic DC subsets. HuNSG mice were injected with polyICLC and euthanized after the indicated time points. PBS-injected mice were sacrificed after 14 hours. Splenocytes were stained for flow cytometry and MFI for CD86 and CD83 is shown for CD1c+ cDCs (top), CD303+ pDCs (middle), and CD141+ cDCs (bottom). (B) Same as in panel A, showing fold up-regulation over the mean MFI of the PBS samples. (C) Time-course for serum cytokine levels in huNSG mice after the injection of polyICLC. HuNSG mice were injected and euthanized as in panel A. Serum cytokine levels were determined for human IL-12p70 (left) and human pan-specific IFN-α (right) by ELISA. Data represent 3 independent experiments. (D) Cytokine levels in the serum of huNSG mice injected with different TLR agonists. HuNSG mice were injected as in Figure 2A. At 11 hours after injection, mice were euthanized, and human IL-12p70 (left) as well as human pan-specific IFN-α (right) were determined in the serum by ELISA. Composite data from 5 independent experiments are shown. Each data point represents one individually analyzed mouse. Statistical analysis was performed with the Mann-Whitney U test.

Figure 4

Depletion of human CD141-positive DCs reduces IFN-α levels after polyICLC stimulation in huNSG mice. (A) CD141+ cDC depletion efficiency in huNSG mice. HuNSG mice were injected with PBS or 10 μg of αClec9A antibody intraperitoneally on 3 consecutive days. At 10 hours after the last injection, mice were injected with polyICLC and euthanized 11 hours later. Splenocytes were stained for flow cytometry, and the percentage of CD141+ cDCs within the CD11c-positive gate was determined. (B) CD1c+ cDC maturation in huNSG mice without and with CD141+ cDC depletion. Same as in panel A with flow cytometry staining for CD86 on CD1c+ cDCs. (C) Serum levels of IFN-α in huNSG mice without and with CD141+ cDC depletion. After injections, as in panel A, serum cytokine levels were determined for human pan-specific IFN-α by ELISA. Left, a representative experiment; right, plot: composite data from 3 experiments. Each data point represents one individually analyzed mouse. Statistical analysis was performed with the Mann-Whitney U test.

Figure 5

IFN-α production by CD141+ cDCs in response to TLR3 ligand. (A) Human cDCs produce IFN-α in response to polyICLC. PBMCs of 3 donors were separated into a cDC and a non-cDC fraction by magnetic-activated cell sorting (MACS) separation. Then, 2 × 105 cells were plated and stimulated for 14 hours with 25 μg/mL polyICLC, 5 μg/mL GLA, or 4 μg/mL R848. Pan IFN-α levels were determined in the cell supernatants after 14 hours by the use of ELISA. (B) IFN-α production by DC subsets after TLR stimulation. PBMCs were sequentially separated into CD141+ cDCs, CD304+ pDCs, and CD1c+ cDCs by MACS separation. A total of 0.25 × 105 cells of the positive fractions as well as the final negative fraction were plated, stimulated, and IFN-α levels were determined as in panel A. (C) Same as in panel B but showing composite data from 3 donors. (D) Intracellular staining for IFN-α in CD141+ cDCs stimulated with polyICLC. cDCs were isolated from PBMCs by MACS separation and stimulated with polyICLC for 9 hours with 10 μg/mL brefeldin A. Cells were stained for surface markers followed by intracellular cytokine staining for IFN-α. Gating was performed as described in Figure 1A, and plots display the CD141+ cDCs. Numbers indicate the percentage of IFN-α producing cells with (right) and without (left) polyICLC stimulation. (E) Fold change of intracellular levels of IFN-α in 4 donors. Same as in panel D but shown for the CD1c+ and CD141+ cDCs fractions. Statistical analysis was performed with a paired t test. (F) IFN-α secretion by DC subsets. Cellular supernatants shown in panel C were run on pan-IFN-α−specific and IFN-α2−specific ELISAs in parallel. The plot shows composite data for the percentage of IFN-α2 of the pan IFN-α amount for 3 donors. Error bars indicate SD. (G) Transcriptional profiles for IFN-α subtypes in CD141+ cDCs. CD141+ cDCs were isolated from 3 donors by MACS and stimulated with polyICLC for 8 hours before RNA isolation. Sybr-green based Q-PCR was performed for the different IFN-α subtypes and transcript numbers were related to 1000 GAPDH copies. Results are displayed as percentage of the indicated IFN-α subtype transcript number in relation to the total IFN-α count. Error bars indicate SD. (H) Same as in panel H but for CD304+ pDCs stimulated with R848 for 1 hour.

Figure 6

αDEC-A647 is taken up most efficiently by CD141+ cDCs in vivo. (A) 5 μg of αDEC-A647 or IgG-A647 was injected intraperitoneally into huNSG mice. After 3 hours, the mice were euthanized, and antibody uptake into the different cellular subsets in the spleen was analyzed by fluorescence-activated cell sorting. (B) Same as in panel A but shown for mice from 2 independent experiments, including 3- and 5-hour time points, which showed similar results. Each data point represents one individually analyzed mouse. Statistical analysis was performed with paired t tests.

Figure 7

Characterization of T-cell responses induced by vaccination with αDEC-205-EBNA1 and polyICLC in huNSG mice. (A) αDEC-205-EBNA1 plus polyICLC vaccination primes specific T cells in huNSG mice. HuNSG mice were vaccinated with 5 μg of IgG-EBNA1 or αDEC-205-EBNA1 with the use of 50 μg of polyICLC as adjuvant and boosted with the same dose of antibodies and adjuvant 4 weeks later. The mice were euthanized 6 to 8 weeks after the boost. T-cell clones were generated by limiting dilution cloning of specific T cells after an IFN-γ capture assay after restimulation with 5μM EBNA1 peptide library for 12 hours. IFN-γ secretion was analyzed by ELISA in the supernatant. (B) The specificity of 3 expanded T-cell clones (c15, c16, c21) are shown after restimulation with 5 different peptide subpools of EBNA1 by measuring IFN-γ secreted into the supernatant. Data are represented as mean ± SD from duplicate or triplicate wells of IFN-γ ELISA. (C) The EBNA1 specific T-cell clone c16 recognizes autologous LCLs. Autologous LCLs (Auto LCL) or allogeneic LCLs (Allo LCL), unmanipulated or loaded for 1 hour with 5μM EBNA1 peptides, were incubated with the expanded EBNA1-specific T-cell clone c16 (E:T=1:2), T-cell activity was determined after 18 hours by measuring released IFN-γ by ELISA. (D) Same as in panel C but analyzing T-cell function by CD107a staining after 6 hours of coculture. (E) The T-cell response of clone c16 is MHC class II-restricted. T-cell clone c16 was exposed to the autologous LCLs in the absence or presence of the indicated HLA blocking antibodies and IFN-γ was measured in the supernatants by ELISA. One representative experiment of 2 is shown. Statistical analysis was performed by Mann-Whitney U tests, and data are represented as mean ± SD.